Bacterial live vector vaccines expressing chromosomally-integrated foreign antigens

ABSTRACT

Bacterial live vector vaccines represent a vaccine development strategy that offers exceptional flexibility. In the present invention, genes encoding protective antigens of unrelated bacterial, viral, parasitic, or fungal pathogens are expressed in an attenuated bacterial vaccine strain that delivers these foreign antigens to the immune system, thereby eliciting relevant immune responses. Rather than expressing these antigens using only low copy expression plasmids, expression of foreign proteins is accomplished using both low copy expression plasmids in conjunction with chromosomal integrations within the same live vector. This strategy compensates for the inherent disadvantage of loss of gene dosage (versus exclusive plasmid-based expression) by integrating antigen expression cassettes into multiple chromosomal sites already inactivated in an attenuated vector.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under Grant NumberAI077911 and Grant Number AI095309 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

SEQUENCE LISTING

A sequence listing in electronic (ASCII text file) format is filed withthis application and incorporated herein by reference. The name of theASCII text file is “2016_0626_ST25.txt”; the file was created on Aug.11, 2016; the size of the file is 92 KB.

TECHNICAL FIELD

The invention generally relates to the provision of live vector vaccinesthat can be used to vaccinate a subject against bacterial, viral orparasitic pathogens. In particular, the invention relates to bacteriallive vector vaccines that express chromosomally-integrated antigenexpression cassettes encoding selected antigens, such as protectiveantigens of unrelated bacterial, viral or parasitic pathogens.

BACKGROUND OF INVENTION

Excellent progress has been made over the past twenty years in theadaptation of attenuated bacterial vaccine strains for expression offoreign antigens to create multivalent live vector vaccines. This hasincluded a devotion of significant effort to the creation of expressiontechnologies which either directly or indirectly address the importantproblem of metabolic stress often associated with expression of foreignimmunogens.[1,2] It is recognized that inappropriate synthesis of highlevels of foreign protein in an effort to induce an antigen-specificprotective immune response can adversely affect the fitness and growthrate of an already attenuated vaccine strain, resulting inover-attenuation and loss of immunity directed at both the live vectorand foreign antigen. If these target immunogens are encoded by multicopyexpression plasmids, these undesirable metabolic fluxes can result inplasmid loss in the absence of selective pressure, which ultimatelydefeats the strategy of live vector-mediated delivery of vaccineantigens.

Effective genetic stabilization systems have been developed forenhancing the retention of multicopy plasmids encoding regulatedsynthesis of foreign antigens, without the further requirement to selectwith antibiotics.[3,4,5] Antigen export systems have also been developedto reduce proteolytic degradation of foreign antigens within thecytoplasm and more effectively deliver these antigens to the immunesystem to enhance immunogenicity. [6,7,8,9] Thus, a variety of genetictechniques and technologies are now available for efficient delivery ofone or more antigens using live vector vaccines. However, significantproblems remain associated with this technology. For example inclusionof more than one gene encoding a foreign antigen of interest within asingle multicopy plasmid can lead to large plasmids which ultimatelyprove to be genetically unstable, reducing both antigen synthesis andthe ensuing immune responses.[10]

Novel strategies for engineering live vector vaccines to express highlevels of a foreign antigen or to express two or more different antigensare needed.

BRIEF SUMMARY OF INVENTION

The present invention is based on a novel strategy of engineering livevector vaccines to have antigen expression cassettes encoding an antigenof interest integrated into two or more different chromosomal locations,and optionally carrying a plasmid-based expression system. Live vectorvaccines engineered in this manner can deliver sufficiently immunogeniclevels of the chromosomally encoded antigens to a subject. The strategicintegration of antigen expression cassettes into multiple locationswithin the chromosome of the selected live vector results in productionof sufficient levels of the encoded antigen, while avoiding adverseeffects on the fitness and growth rate of the vector.

In a first embodiment, the invention is directed to an attenuated strainof Salmonella enterica serovar Typhi (hereinafter “S. Typhi”) havingdisruptions of two or more chromosomal locations selected from the groupconsisting of the guaBA locus, the htrA locus, the clyA locus, the rpoSlocus, and the ssb locus. In one aspect of this embodiment, theattenuated strain of S. Typhi is the strain CVD 910 which hasdisruptions of the guaBA locus, the htrA locus, and the rpoS locus.

In a second embodiment, the invention is directed to an antigen-encodingattenuated strain of S. Typhi, wherein the strain comprises:

(a) disruptions of two or more chromosomal locations, wherein thechromosomal locations are selected from the group consisting of theguaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssblocus, and

(b) chromosomal-based expression systems integrated into the locationsof the chromosomal disruptions, wherein each chromosomal-basedexpression system comprises an antigen expression cassette encoding anantigen of interest.

In one aspect of this embodiment, the antigen of interest is aprotective antigen of, for example, an unrelated bacterial, viral,parasitic, or fungal pathogen. In a particular aspect, the antigen ofinterest is one or more of the cell binding domain of C. difficile toxinA (CBD/A), the cell binding domain of C. difficile toxin B (CBD/B), thecell binding domain of C. difficile binary toxin (BT), the LcrV antigenof Yersinia pestis and the capsular F1 antigen of Yersinia pestis. In afurther aspect, each chromosomal-based expression system comprises anantigen expression cassette encoding a different antigen of interest.

In another aspect of this embodiment, the antigen-encoding attenuatedstrain of S. Typhi is the strain CVD 910 which has disruptions of theguaBA locus, the htrA locus, and the rpoS locus, and which has achromosomal-based expression system integrated into each site ofdisruption that encodes one or more antigens of interest. In aparticular aspect, the antigen-encoding attenuated strain of S. Typhi isthe strain CVD 910-3A which has disruptions of the guaBA locus, the htrAlocus, and the rpoS locus, and which comprises antigen expressioncassettes integrated into the locations of chromosomal disruption,wherein each antigen expression cassette encodes the cell binding domainof C. difficile toxin A.

In a third embodiment, the invention is directed to an antigen-encodingattenuated strain of S. Typhi, wherein the strain comprises:

(a) disruptions of two or more chromosomal locations, wherein thechromosomal locations are selected from the group consisting of theguaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssblocus,

(b) chromosomal-based expression systems integrated into the locationsof the chromosomal disruptions, wherein each chromosomal-basedexpression system comprises an antigen expression cassette encoding anantigen of interest, and

(c) one or more plasmid-based expression systems, wherein eachplasmid-based expression system encodes an antigen of interest.

In one aspect of this embodiment, the antigens of interest areindividually protective antigens of, for example, unrelated bacterial,viral or parasitic pathogens. In a particular aspect, the antigens ofinterest are individually one or more of the cell binding domain of C.difficile toxin A (CBD/A), the cell binding domain of C. difficile toxinB (CBD/B), the cell binding domain of C. difficile binary toxin (BT),the LcrV antigen of Yersinia pestis and the capsular F1 antigen ofYersinia pestis. In a further aspect, the antigens of interest aredifferent.

In another aspect of this embodiment, the antigen-encoding attenuatedstrain of S. Typhi is the strain CVD 910-3A which has disruptions of theguaBA locus, the htrA locus, and the rpoS locus, and which comprisesantigen expression cassettes integrated into the locations ofchromosomal disruption, wherein each antigen expression cassette encodesthe cell binding domain of C. difficile toxin A, a further disruption inthe ssb locus, and which has an SSB-stabilized plasmid-based expressionsystem. In a particular aspect, the antigen-encoding attenuated strainof S. Typhi is the strain CVD 910-3Assb(pSEC10-CBD/B) which hasdisruptions of the guaBA locus, the htrA locus, and the rpoS locus, andwhich comprises antigen expression cassettes integrated into thelocations of chromosomal disruption, wherein each antigen expressioncassette encodes the cell binding domain of C. difficile toxin A, afurther chromosomal deletion of the ssb locus, and an SSB-stabilizedplasmid-based expression system encoding the cell binding domain of C.difficile toxin B.

In a fourth embodiment, the invention is directed to an antigen-encodingattenuated strain of S. Typhi, wherein the strain comprises:

(a) disruptions of four chromosomal locations, wherein the chromosomallocations are selected from the group consisting of the guaBA locus, thehtrA locus, the clyA locus, and the rpoS locus,

(b) chromosomal-based expression systems integrated into the locationsof the chromosomal disruptions, wherein each chromosomal-basedexpression system comprises an antigen expression cassette encoding anantigen of interest, and

(c) a plasmid-based expression system, wherein the plasmid-basedexpression system encodes an antigen of interest.

The antigens of interest may be the same or different in a singlestrain, and a single copy or multiple copies of the same antigen can beexpressed in a single strain. In one aspect of this embodiment, theantigens of interest are protective antigens of, for example, unrelatedbacterial, viral or parasitic pathogens. In a particular aspect, theantigens of interest are one or more of the cell binding domain of C.difficile toxin A (CBD/A), the cell binding domain of C. difficile toxinB (CBD/B), the cell binding domain of C. difficile binary toxin (BT),the LcrV antigen of Yersinia pestis and the capsular F1 antigen ofYersinia pestis. In a further aspect, the attenuated strain of S. Typhiexpresses three different antigens of interest.

In another aspect of this embodiment, the antigen-encoding attenuatedstrain of S. Typhi is the strain CVD 910-3A which has disruptions of theguaBA locus, the htrA locus, the rpoS locus and the clyA locus, andwhich comprises antigen expression cassettes integrated into thelocations of chromosomal disruption, wherein at least one of the antigenexpression cassettes encodes the cell binding domain of C. difficiletoxin A (CBD/A), wherein at least one of the antigen expressioncassettes encodes the binary toxin (BT) of C. difficile, which has afurther disruption in the ssb locus, and which has an SSB-stabilizedplasmid-based expression system expressing the cell binding domain of C.difficile toxin B (CBD/B). In a particular embodiment, three of theantigen expression cassettes encode the cell binding domain of C.difficile toxin A (CBD/A) and one of the antigen expression cassettesencodes the binary toxin (BT) of C. difficile. In a further particularembodiment, two of the antigen expression cassettes encode the cellbinding domain of C. difficile toxin A (CBD/A) and two of the antigenexpression cassettes encode the binary toxin (BT) of C. difficile.

In a particular aspect, the antigen-encoding attenuated strain of S.Typhi is the strain CVD 910-3A-GB2ssb(pSEC10-CBD/B) which comprises (i)disruptions of the guaBA locus, the htrA locus, the rpoS locus and theclyA locus, and which comprises antigen expression cassettes integratedinto the locations of chromosomal disruption, wherein antigen expressioncassette located in guaBA chromosomal disruption encodes the binarytoxin (BT) of C. difficile, wherein antigen expression cassettes locatedin htrA, rpoS and clyA chromosomal disruptions encode the cell bindingdomain of C. difficile toxin A (CBD/A), (ii) a disruption in the ssblocus, and (iii) an SSB-stabilized plasmid-based expression systemencoding the cell binding domain of C. difficile toxin B (CBD/B.

In a fifth embodiment, the invention is directed to a live vectorvaccine comprising an antigen-encoding attenuated strain of S. Typhi asdefined herein, and a pharmaceutically-acceptable carrier or diluent.

In a sixth embodiment, the invention is directed to methods of inducingan immune response to an antigen of interest in a subject, comprisingadministering to a subject an antigen-encoding live vector vaccine asdefined herein that expresses an antigen of interest.

In a seventh embodiment, the invention is directed to methods ofvaccinating a subject with a protective antigen, comprisingadministering to a subject an antigen-encoding live vector vaccine asdefined herein that expresses a protective antigen.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedherein, which form the subject of the claims of the invention. It shouldbe appreciated by those skilled in the art that any conception andspecific embodiment disclosed herein may be readily utilized as a basisfor modifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of use, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thatany description, figure, example, etc. is provided for the purpose ofillustration and description only and is by no means intended to definethe limits the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Schematic depiction of the strategy for chromosomal integrationof the antigen expression cassette P_(ompC)-gfpuv, encoding the modelfluorescent antigen GFPuv. An osmotically-controlled GFPuv-encodingcassette (tandem white circle and hatched thick arrow) was constructedand linked to an aph marker encoding resistance to kanamycin (shadedthick arrow), flanked by FRT recombination sites (black triangles). Theincoming P_(ompC)-gfpuv-aph cassette was integrated into the live vectorchromosome using the λ Red recombination system, followed by removal ofthe aph marker using FLP recombinase, to yield the final live vectorstrain bearing no genes encoding resistance to antibiotics. Thebacterial chromosome is represented by 5′-proximal and 3′-terminaldarkened rectangles, and the black circle labeled with a “P” representsthe wild-type chromosomally-encoded promoter of the deleted target openreading frame (e.g., guaBA or htrA).

FIG. 2. Flow cytometry histograms of GFPuv-mediated fluorescence encodedby P_(ompC)-gfpuv gene cassettes integrated into either the guaBA (thicksolid line), htrA (thin hatched line), or clyA (thick broken line) sitesof the attenuated S. Typhi live vector vaccine candidate CVD 910,compared to the vaccine strain alone (thin dotted line). Fluorescenceintensities are measured for individual bacterial cells grown underinducing conditions of 200 mM NaCl in rich medium at 37° C./250 rpm for16 hr.

FIG. 3. Schematic depiction of the strategy for chromosomal integrationof the cell binding domain from toxin A of C. difficile. A syntheticcodon-optimized gene cassette encoding the cell binding domain fromtoxin A designated 14cbd/a was prepared where the osmotically regulatedP_(ompC) promoter was genetically fused to a promoterless 14cbd/a gene(tandem white circle and hatched thick arrow) and linked to an aphmarker encoding resistance to kanamycin (shaded thick arrow), flanked byFRT recombination sites (black triangles). The incomingP_(ompC)-14cbd/a-aph cassette was integrated into the live vectorchromosome using the λ Red recombination system, followed by removal ofthe aph marker using FLP recombinase, to yield the final live vectorstrain bearing no genes encoding resistance to antibiotics. Thebacterial chromosome is represented by 5′-proximal and 3′-terminaldarkened rectangles, and the black circle labeled with a “P” representsthe wild-type chromosomally-encoded promoter of the deleted target openreading frame (e.g., guaBA, htrA or rpoS).

FIG. 4. Western immunoblot analysis. Six hour liquid broth cultures ofCVD 910-2A (“2A”) were compared to cultures of CVD 910-3A (“3A”) undereither inducing (200 mM NaCl to activate P_(ompC)) or non-inducing (15mM NaCl) conditions.

FIG. 5. Schematical depiction of live vaccine strain CVD910-3Assb(pSEC10-CBD/B).

FIG. 6. Comparison of hemolytic activity of fusion proteins expressed inCVD 910-4A, CVD 910-3A-GB2 and CVD 910-4Assb(pSEC10S2-B2). The notedstrains were grown on trypticase soy agar with 5% sheep red blood cellsunder conditions of incubation at 37° C. for 24 hours. The plates werethen photographed without magnification.

FIG. 7. Schematical depiction of live vaccine strain CVD 910-3A-GB2ssb(pSEC10-CBD/B) which contains insertions into the guaBA, htrA, rpoS andclyA loci and carries the plasmid pSEC10-CBD/B.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found, for example, in Benjamin Lewin, Genes VII, published by OxfordUniversity Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technicalreferences.

As used herein, “a” or “an” may mean one or more. As used herein whenused in conjunction with the word “comprising,” the words “a” or “an”may mean one or more than one. As used herein “another” may mean atleast a second or more. Furthermore, unless otherwise required bycontext, singular terms include pluralities and plural terms include thesingular.

As used herein, “about” refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearestsignificant figure.

II. The Present Invention

Live vectors engineered for delivery of foreign antigens to the hostimmune system have performed well in experimental animal models, buthave been only modestly successful in clinical trials. [20] Given theadvances in the development of powerful plasmid-based expressiontechnologies designed to deliver ample levels of foreign protein, it isunlikely that the lack of antigen-specific immunity observed in clinicaltrials is due to insufficient antigen synthesis following immunization.To the contrary, it is likely that inappropriate antigen synthesisoccurring in vivo results in sufficient shock to the metabolism of thelive vector to over-attenuate the strain and destroy immunogenicity.Although various attempts have been made to control the timing offoreign protein synthesis, using tightly regulated promoters to controltranscription of genes in response to host environmental signals forexample, improved immunogenicity in animals has not translated intoimprovements in clinical trials.[12,21,22]

A novel and elegant solution to this dilemma is presented here, whereinover-attenuation is circumvented by linking antigen synthesis to thegrowth rate of the live vector vaccine, such that synthesis is initiallylow after immunization, but steadily increases as the vaccine strainadjusts to prevailing environmental conditions and undergoes limitedreplication within the host. This expression strategy allows forefficient expression of one or even multiple foreign antigens within asingle live vector vaccine strain. It can also be used in conjunctionwith plasmid-based methods by distributing the location of antigenexpression cassettes between the chromosome and an expression plasmid.The approach presented herein thus offers the flexibility ofindependently adjusting the copy number of potentially toxic foreigngenes by integrating a designated number of copies into the chromosome.By appropriate integration of foreign genes into chromosomal loci whoseinduction of expression is intimately associated with the physiology andgrowth rate of the vaccine strain, it becomes possible to “tune” foreignantigen synthesis to the metabolic state of the live vector.

The present invention is therefore based on the discovery that deliveryof sufficiently immunogenic levels of chromosomally-encoded antigens toa subject can be accomplished through strategic integration of antigenexpression cassettes into multiple locations within the live vectorchromosome, thereby compensating for loss of copy number afforded bysystems using stable low copy plasmids, while avoiding furtherattenuation of the vaccine strain. Integration of multiple cassettesalso avoids the need for strong constitutive promoters to enhanceantigen synthesis from a single gene copy, an approach which does notnecessarily lead to adequate antigen synthesis or immuneresponses.[11,12]

The present invention is directed to several related embodiments,including (i) an attenuated strain of S. Typhi having disruptions of twoor more chromosomal locations selected from the group consisting of theguaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssblocus, and (ii) antigen-expressing attenuated strains of S. Typhi havinga chromosomal-based expression system which comprises an antigenexpression cassette integrated into two or more locations of chromosomaldisruptions, (iii) antigen-expressing attenuated strains of S. Typhihaving a chromosomal-based expression system which comprises an antigenexpression cassette integrated into two or more locations of chromosomaldisruptions as well as a plasmid-based expression system, (iv) a livevector vaccine comprising an antigen-expressing attenuated strain of S.Typhi as defined herein, and a pharmaceutically-acceptable carrier ordiluent, (v) methods of inducing immune responses to an antigen ofinterest in a subject using the live vector vaccines as defined herein,and (vi) methods of vaccinating a subject using the live vector vaccinesas defined herein.

Attenuated Strains of S. Typhi

As suggested above, in one embodiment the present invention is directedto attenuated strains of Salmonella enterica serovar Typhi havingdisruptions of two or more chromosomal locations. S. Typhi is awell-tolerated live vector that can deliver multiple unrelatedimmunogenic antigens to the human immune system. S. Typhi live vectorshave been shown to elicit antibodies and a cellular immune response toan expressed antigen. S. Typhi is characterized by enteric routes ofinfection, a quality which permits oral vaccine delivery. S. Typhi alsoinfects monocytes and macrophages and can therefore target antigens toprofessional APCs.

The genetic disruptions are sufficiently extensive to ensure that activeforms of the protein(s) encoded by the locus harboring the disruptionare not produced by the bacteria. While the skilled artisan willunderstand that the characteristics and scope of the disruptions canvary widely, in one non-limiting aspect the disruptions are sufficientlyextensive to ensure that neither the protein(s) encoded by the locus,nor fragments thereof, can be detected in bacteria having thedisruptions.

The skilled artisan will recognize that strains of bacteria having thedisruptions can be readily produced via several techniques known in theart, including the λ Red-mediated site-directed mutagenesis method. [16]Other, less efficient, chromosomal deletion/integration technologiesused in the past involve the use of suicide plasmids. These suicideplasmids exhibit replication which is exclusively dependent on the pirprotein; successful deletions/integrations are dependent onrecA-mediated homologous chromosomal crossovers, and counter-selectionwith sacB. [29]

The chromosomal locations having the two or more disruptions are theguaBA locus, the htrA locus, the clyA locus, the rpoS locus, and the ssblocus. The disruptions of these loci can be disruptions of theendogenous coding sequences, non-coding control sequence, promotersequences, or a combination thereof. In the case of the guaBA locus, forexample, the coding region can be disrupted without damaging thepromoter sequence for the loci.

The chromosomal disruptions can include any combination of deletions orinsertions of sequences comprising the disrupted loci.

The attenuated strains of S. Typhi may have disruptions in anycombination of two, three or four of the loci and sites, or even allfive of the loci.

Specific examples of such attenuated strains of S. Typhi includingstrain CVD 910, which contains disruptions in the guaBA locus and thehtrA locus. Because the parent Ty2 strain used in the production of CVD910 has the rpoS locus naturally inactivated, CVD 910 also contains adisruption of the rpoS locus. Thus, CVD 910 contains disruptions inthree chromosomal locations: the guaBA locus, the htrA locus, and therpoS locus.

Antigen-Encoding Attenuated Strains of S. Typhi

As suggested above, in a related embodiment the invention is directed toantigen-encoding attenuated strains of S. Typhi having achromosomal-based expression system integrated into two or more of thedisrupted chromosomal locations within the bacteria. Thus, the inventionincludes the attenuated strains of S. Typhi as defined herein, includingstrain CVD 910, which have been engineered to have antigen expressioncassettes integrated into the locations of chromosomal disruptions.

The chromosomal-based expression systems used in the antigen-encodingattenuated strains of S. Typhi are simple in nature in that theycomprises a genetic cassette (antigen expression cassette) comprisingthe coding sequence of an antigen of interest and, optionally, anexogenous promoter to direct transcription of the coding sequence. Insome circumstances and depending on the identity of the coding sequenceand/or promoter, additional 5′ and/or 3′ non-coding sequence associatedwith the coding sequence of the antigen of interest can be included inthe cassette. Together, these sequences make up an antigen expressioncassette that can be inserted into one or more disrupted bacterialchromosomes. Because the attenuated strains of S. Typhi defined hereinhave at least two chromosomal disruptions, antigen expression cassettesencoding different antigens can be used in the same strain of bacteria.In those strains of S. Typhi having three chromosomal disruptions, up tothree different antigens may be expressed, with up to four differentantigens in those strains of S. Typhi having four chromosomaldisruptions, and up to five different antigens in those strains of S.Typhi having five chromosomal disruptions. The skilled artisan will alsorecognize that different combinations of antigens can be expressed in agiven strain depending on the number of disruptions and the selectedantigen expression cassettes. For example, where a strain has threedisruptions, the same antigen expression cassette (encoding antigen A,for example) can be inserted into each of the three sites.Alternatively, an antigen expression cassette encoding antigen A couldbe inserted into two of the sites, while an antigen expression cassetteencoding antigen B could be inserted into the third site. In a furtheralternative, an antigen expression cassette encoding antigen A could beinserted into one of the sites, an antigen expression cassette encodingantigen B could be inserted into the second site, and an antigenexpression cassette encoding antigen C could be inserted into the thirdsite. Thus, the identity of the antigen encoded by each antigenexpression cassette is individually selected and any combination ofantigens may be used. This expressly includes instances where aparticular antigen is encoded by two or more cassettes as well asinstances where a particular antigen is encoded by only one cassette.

Antigens

The antigen expression cassettes of the present invention preferablyexpress an antigen for presentation to a host to elicit an immuneresponse resulting in immunization and protection from disease. Theantigens of interest that may be expressed in the attenuated strains ofS. Typhi of the present invention are unlimited in identity and include,but are not limited to, antigens from foreign bacteria (e.g., proteinsnot already expressed by S. Typhi), viruses, and parasitic organisms.Exemplary antigens are protective antigens (i.e., an antigen that whenbound by an antibody, result in the death of the organism expressing theantigen). Because the attenuated strains of S. Typhi may be used as livevector vaccines, one practicing the invention will be motivated, in oneaspect, to use antigens suspected of or known to induce a protectiveimmune response in a subject. As another example, antigens suspected ofor known to induce an active immune response in a subject topre-existing infection may also be used.

Exemplary antigens that can be used to induce a protective immuneresponse include detoxified versions of enterotoxin A (TcdA),enterotoxin B (TcdB), and binary toxin (transferase; Cdt) of Clostridiumdifficile, and fragments thereof, such as the cell-binding domains ofTcdA and TcdB, and the binding domain of Cdt (CdtB). These antigens canbe used to produce a mono-, bi- or multivalent live vector vaccine thatin turn can be used to vaccinate a subject against infections caused byC. difficile. With the ability to impart immunity that recognizes bothtoxins and putative colonization factors, a subject can be vaccinatedagainst disease that occurs at two critical stages ofinfection—colonization and toxin production.

Additional exemplary antigens include the LcrV antigen and the capsularF1 antigen of Yersinia pestis, which are required for secretion ofvirulence effectors proteins and are virulence factors themselves.Additional Yersinia pestis antigens include pH 6 antigen (Psa), aputative colonization factor, and Yop B/YopD, two essential proteinscomprising the translocon region of the type 3 secretion (T3SS) needle.

Given the ease with which antigen expression cassettes can be produced,and the straightforwardness of inserting and removing the cassettes fromlocations of chromosomal disruptions in bacteria, it will be clear tothe skilled artisan that a very wide range of different antigens can beexpressed using the chromosomal-based expression systems of theattenuated strains of S. Typhi defined herein. These same antigens canalso be expressed in the bacteria using the plasmid-based expressionsystems discussed below.

Plasmid-Based Expression Systems

In a related embodiment, the invention is also directed toantigen-encoding attenuated strains of S. Typhi having thechromosomal-based expression system discussed above, as well as aplasmid-based expression system. The inclusion of a plasmid-basedexpression system within the bacteria provides additional flexibilityfor expressing antigens of interest in the attenuated strains of S.Typhi of the invention. The plasmid-based expression system allowsexpression of additional copies of an antigen expression cassette thatis integrated into the bacterial chromosome or an antigen expressioncassette encoding an antigen of different identity. Thus, as definedherein the antigen expression cassettes can be inserted to both thelocations of chromosomal disruptions (in terms of the chromosomal-basedexpression systems) and expression plasmids (in terms of theplasmid-based expression systems).

SSB-Stabilized Plasmid-Based Expression System

Examples include the plasmid-based expression systems disclosed in U.S.Pat. Nos. 6,703,233, 6,969,513, 6,977,176, 7,141,408, 7,125,720 and7,138,112, each of which is incorporated herein by reference in theirentirety. The systems described in these patents are multicopyexpression plasmids into which plasmid maintenance systems have beenincorporated. Such multicopy expression plasmids produce a gene dosageeffect which enhances the level of expression of the antigen ofinterest. In a specific example, a plasmid-based expression system hasbeen developed in which a low copy expression plasmid has beenengineered to encode an essential single-stranded binding protein (SSB)which has been deleted from the bacterial vaccine strain. Since SSB isessential for DNA replication, recombination, and repair, ssb-deletedbacteria must maintain the expression plasmid to enable survival.Therefore, if SSB-stabilized plasmids are used to encode one or moreforeign antigens, then bacteria become committed to foreign antigensynthesis. If expression of foreign antigens in the cytoplasm of thebacteria becomes toxic, antigen export systems can be further introducedinto these SSB-stabilized plasmids to export fusion proteins out of thecytoplasm and minimize metabolic disruption.

ClyA Fusion Protein Plasmid-Based Expression Systems

A further example of a suitable plasmid-based expression system isdisclosed in WO 09/149083, incorporated herein by reference in itsentirety, which makes use of the S. Typhi HlyE family of exportproteins, including the cryptic hemolysin (ClyA), encoded by thecytolysin A gene (clyA). ClyA from S. Typhi was first described byWallace et al. who also reported the crystal structure for thehomologous hemolysin from E. coli. [26] This hemolysin has beendescribed previously and variously referred to as ClyA, HlyE, or SheA.

The crystal structure of ClyA in E. coli has been resolved. [26] Theunique structure can be roughly divided into several domains, a headdomain, a body domain and a tail domain. The body domain consists of abundle of helixes (A, B, C, D, F). The tail domain is a helix G whichextends to half the length of the body. The head domain consists of ashort β hairpin (β-tongue) and two small helices (D and E), eachflanking the β-tongue. It was suggested that the β-tongue might becritical for pore formation and hence for the hemolytic activity. [26]Through site directed mutagenesis, it was found that many regions ofClyA were important for the hemolytic activity. [27]

The ClyA protein is exported from both E. coli and S. Typhi and it iscapable of exporting passenger proteins (and antigens of interest) thathave been genetically fused to the 3′-terminus of the clyA open readingframe. It is demonstrated that the proper folding of these fusionproteins occurs such that the inherent biological activity of thedomains involved is maintained.

The amino acid and nucleotide sequence for the isolated S. Typhi clyAgene and ClyA protein (from Salmonella serovar Typhi strain Ty2) areprovided as SEQ ID NO:39 and SEQ ID NO:38, respectively. A syntheticcodon-optimized version of the S. Typhi clyA gene, as described andutilized herein, is provided in SEQ ID NO:40. Other HlyE family membersthat may be utilized as export proteins herein are also available andknown to those of ordinary skill in the art. The family members includea second S. Typhi cytolysin A (the clyA gene is set forth in SEQ IDNO:41 and it is available under GENBANK Accession No. AJ313034);Salmonella paratyphi cytolysin A (the clyA gene sequence for cytolysin Ais set forth in SEQ ID NO:42 and it is available under GENBANK AccessionNo. AJ313033); Shigella flexneri truncated HlyE (the hlyE gene sequenceis set forth in SEQ ID NO:43 and it is available under GENBANK AccessionNo. AF200955); Escherichia coli HlyE (the hlyE gene sequence is setforth in SEQ ID NO:44 and it is available under GENBANK Accession No.AJ001829).

As indicated above, the HlyE family of proteins typically causescytolysis of target cells, including hemolysis of erythrocytes. Becausecytolysins/hemolysins may be considered to be virulence factors, thepresent invention encompasses the use of variants of HlyE family membersthat have been mutated such that they lack, or have reduced, hemolyticactivity. The ability of these variants to be exported from a bacterialcell producing them, alone or in the context of fusion to a protein ofinterest, has been maintained. Thus, the non-hemolytic variants of HlyEfamily members have reduced or no hemolytic activity, and yet are fullyfunctional in the plasmid-based expression systems of the presentinvention. Such variants include the S. Typhi cytolysin A (ClyA) proteinof SEQ ID NO:38 having a single mutation selected from the groupconsisting of an 5195N mutation, an I198N mutation, an A199D mutation,an E204K mutation, and a C285W mutation; an I198N, C285W doublemutation; and an I198N, A199D, E204K triple mutation. The S. Typhicytolysin A (ClyA) protein may also have the amino acid sequence setforth in SEQ ID NO:38 and a C285W mutation, as well as one additionalmutation selected from the group consisting of an I198N mutation, anA199D mutation, and an E204K mutation.

The plasmid-based expression systems comprising ClyA fusion proteinsdescribed herein can be used to express and export a wide variety offusion proteins comprising an export protein and an antigen of interest.The export protein::antigen of interest fusion protein construct ispresent in an antigen expression cassette, which in turn is present inan expression plasmid to facilitate the recombinant production of theprotein of interest. Typically the expression plasmid will comprise anorigin of replication and other structural features that control andregulate the maintenance of the expression plasmid in the host cell.Exemplary expression plasmids are well known to the skilledartisan.[7,23,28] The key aspect of such expression plasmids is copynumber, which can range from several hundred per chromosomal equivalentto one per chromosomal equivalent. Preferably the copy number of theexpression plasmids is between 5 and 15 copies per chromosomalequivalent.

Live Vector Vaccines

As suggested above, and in a related embodiment, the invention isdirected to a live vector vaccine comprising one or more of theantigen-encoding attenuated strains of S. Typhi as defined herein, and apharmaceutically-acceptable carrier or diluent.

It is contemplated that the live vector vaccines of the presentinvention will be administered as pharmaceutical formulations for use invaccination of individuals, preferably humans. In addition to thestrains of S. Typhi, the vaccines will thus includepharmaceutically-acceptable carriers, and optionally, may include othertherapeutic ingredients, such as various adjuvants known in the art.

The carrier or carriers must be pharmaceutically acceptable in the sensethat they are compatible with the therapeutic ingredients and are notunduly deleterious to the recipient thereof. The therapeutic ingredientor ingredients are provided in an amount and frequency necessary toachieve the desired immunological effect.

The mode of administration and dosage forms will affect the therapeuticamounts of the compounds which are desirable and efficacious for thevaccination application. However, the live vector vaccines are deliveredin an amount capable of eliciting an immune reaction in which it iseffective to increase the subject's immune response to the antigen(s) ofinterest. An immunogenic amount is an amount which confers an increasedability to prevent, delay or reduce the severity of the onset of adisease, as compared to such abilities in the absence of suchimmunization. It will be readily apparent to one of skill in the artthat this amount will vary based on factors such as the weight andhealth of the recipient, the type of antigen(s) being expressed, thetype of infecting organism being combatted, and the mode ofadministration of the vaccines.

The vaccines may be formulated for any suitable means and/or methods fordelivering the live vector vaccines to a corporeal locus of the subjectwhere the live vector vaccines are intended to be effective intriggering an immune response, for example, for oral, sublingual,intranasal, intraocular, rectal, transdermal, mucosal, pulmonary,topical or parenteral administration. Parenteral modes of administrationinclude without limitation, intradermal, subcutaneous (s.c., s.q.,sub-Q, Hypo), and intramuscular (i.m.). Any known device useful forparenteral injection or infusion of vaccine formulations can be used toeffect such administration. In preferred aspects of each of theembodiments on the invention, the vaccines are administered to a subjectas an oral formulation, in particular, to the oral mucosa.

The dose rate and suitable dosage forms for the live vector vaccines ofthe present invention may be readily determined by those of ordinaryskill in the art without undue experimentation, by use of conventionalantibody titer determination techniques and conventionalbioefficacy/biocompatibility protocols. Among other things, the doserate and suitable dosage forms depend on the particular antigenemployed, the desired therapeutic effect, and the desired time span ofbioactivity.

Formulations of the vaccines can be presented, for example, as discreteunits such as capsules, cachets, tablets or lozenges, each containing apredetermined amount of the vaccine; or as a suspension.

Depending on the means of administration, the vaccines may beadministered all at once, such as with an oral formulation in a capsuleor liquid, or slowly over a period of time, such as with anintramuscular or intravenous administration. The vaccines may also beadministered to the subject more than once, as boosters, for example,where administration of separate doses of the vaccines may be separatedin time by hours, days, weeks or months.

In each embodiment and aspect of the invention, the subject is a human,a non-human primate, bird, horse, cow, goat, sheep, a companion animal,such as a dog, cat or rodent, or other mammal.

Manners of Use

As indicated above, it is intended that the antigen-encoding attenuatedstrains of S. Typhi defined herein will be grown under conditions thatmay induce expression of the antigens of interest prior to immunization,and formulated as a live vector vaccine for administration to a subject,whereupon an immune response to the antigens of interest, inter alia,will be induced in the subject.

The invention therefore includes methods of inducing an immune responseto an antigen of interest in a subject, comprising administering to asubject a live vector vaccine as defined herein and that expresses anantigen of interest. The invention also includes methods of vaccinatinga subject with a protective antigen, comprising administering to asubject a live vector vaccine as defined herein that expresses aprotective antigen.

The methods contemplate and include administering the live vectorvaccine to the subject only once, or more than once, such as 2, 3, 4, 5or more times.

A non-limiting example of the manner in which the vaccines may be usedincludes use of the vaccine as a nosocomial oral vaccine, administeredto patients seven days after antibiotic treatment for Clostridiumdifficile infection (CDI) to block recurrent disease by eliciting avigorous and rapid anamnestic response in patients primed by the initialC. difficile infection.

IV. Examples Materials and Methods

Bacterial Strains and Culture Conditions.

The attenuated S. enterica serovar Typhi (S. Typhi) live vector vaccinestrain CVD 910 used in these studies is an auxotrophic derivative ofwild-type strain Ty2, with deletions in guaBA and htrA. To improve theclinical acceptability of the live vector vaccine strains, all geneticand bacteriologic manipulations of the live vectors were performed usingan animal product-free medium equivalent to Luria-Bertani medium,comprised of 10 g/liter of Soytone (Teknova; S9052), 5 g/liter Hy-Yest412 (Sigma; Y1001), and 3 g/liter NaCl (American Bioanalytical;AB01915), supplemented with 0.002% guanine (Sigma; G6779).

Construction of Chromosomal Integrations.

Deletion cassettes were constructed for use with the λ Red-mediatedsite-directed mutagenesis method [16] to delete either guaBA, htrA, orclyA from wild-type S. Typhi Ty2. Cassettes encoding upstream anddownstream flanking chromosomal sequences were constructed using primerpairs listed in Table 1 and purified chromosomal DNA from Ty2 as thetemplate DNA.

TABLE 1Primers used in the construction and testing of live vector strains expressingchromosomally-encoded GFPuv. Primer (SEQ ID NO:) Sequence^(a) 5guaBA-for5′- GAATTCTAGCTGCTCATACTTCTGCTGCA -3′ SEQ ID NO: 1 5guaBA-rev5′- GCTAGCCAATTGGGGCAATATCTCACCTGG -3′ SEQ ID NO: 2 3guaBA-for5′- GGATCCACTAGTGTCGATAACCCTTCCTGTGT -3′ SEQ ID NO: 3 3guaBA-rev5′- CTCGAGACAGCACCTACAAGTCTGGCATG -3′ SEQ ID NO: 4 guaBA PCR-for5′- GCGCTGACCACCGGAATACGGCTG -3′ SEQ ID NO: 5 guaBA PCR-rev5′- CATGGCATGGATGAGGCAACCGCGAAGC -3′ SEQ ID NO: 6 5htrA-for5′- GAATTCGTACCTTCAATCAGGCGTTACTGGAAGATG -3′ SEQ ID NO: 7 5htrA-rev5′- GCTAGCCAATTGCGATTAACAGGTAACGCAAAATTGCTGTGTACGTCAG -3′ SEQ ID NO: 83htrA-for 5′- GGATCCACTAGTCTGCGTAAGATTCTCGACAGCAAGCCGTCGGT -3′SEQ ID NO: 9 3htrA-rev5′- CTCGAGCCAGCATCATTTCGGCAGTCATACACACCAGTTCGC -3′ SEQ ID NO: 10htrA PCR-for 5′- GTGTCGCCGATCTTGAAGACGCGGTAGAG -3′ SEQ ID NO: 11htrA PCR-rev 5′- CTATCGACGCCAAGCTGGCCGCTGTCGAC -3′ SEQ ID NO: 125clyA-for 5′- TAGTAATGAGAATTCGCTGGTATTGATCGGCTCTCCGGTAGAGATTAGCGA -3′SEQ ID NO: 13 5clyA-rev5′- GCTAGCCAATTGTGCCTCTTTAAATATATAAATTGCAATTAAGTACCTG -3′ SEQ ID NO: 143clyA-for 5′- GGATCCACTAGTGATACATTTTCATTCGATCTGTGTACTTTTAACGCCCGATSEQ ID NO: 15 AGCG -3′ 3clyA-rev5′- TGATAGTAACTCGAGACAATCCATAAGAAAGGTCAGGCACACTGGGAAGG SEQ ID NO: 16CGACATC -3′ clyA PCR-for 5′- CATGATGGTATCCAGTATGGCACAAGC -3′SEQ ID NO: 17 clyA PCR-rev 5′- GTAATCGACAACATGCTACATCCATCG -3′SEQ ID NO: 18 5FRT-aph-for 5′- GAATTCGCTAGCGCTGGAGCTGCTTCGAAGTTC -3′SEQ ID NO: 19 3FRT-aph-rev 5′- CTCGAGTTCCGGGGATCCGTCGACCTGCAGTTC -3′SEQ ID NO: 20 5gfpuv 5′- CAATTGTGTGGTAGCACAGAATAATGAAAAGT -3′SEQ ID NO: 21 3gfpuv 5′- GCTAGCTCATTATTTGTAGAGCTCATCCAT -3′SEQ ID NO: 22 ^(a)Relevant restriction sites are underlined.

These cassettes were used to exchange chromosomal targets with a Tn5neomycin phosphotransferase cassette (aph), encoding resistance tokanamycin, and recombined into the chromosome using the λ Redrecombination system encoded by pKD46. Final removal of the kanamycinresistance cassette was accomplished using FLP recombinase encoded bypCP20. The integrity of the intended chromosomal deletion mutations wasconfirmed by DNA sequence analysis of the chromosomal locus from eachstrain using PCR primers listed in Table 3. For chromosomal expressionof GFPuv, an antigen expression cassette in which an osmoticallyregulated ompC promoter (P_(ompC) [23]) was linked to gfpuv was selectedand inserted 5′-proximal to the aph resistance marker of chromosomaldeletion cassettes. As shown in FIG. 1, care was taken to preserve thenatural chromosomal promoters controlling transcription of chromosomallyencoded targets, with the intent that synthesis of GFPuv wouldultimately be controlled both by osmolarity (via P_(ompC)) as well asgrowth rate in the case of the guaBA locus,[15] heat shock/environmentalstress in the case of the htrA locus,[18] or possibly low pH for clyA.[19]

Flow Cytometry.

GFPuv-expressing strains were grown overnight at 37° C. on rich solidmedium supplemented with guanine. 2-3 fluorescing colonies were theninoculated into 20 ml of supplemented liquid medium and incubated withshaking at 250 rpm overnight at 37° C. Overnight starter cultures werethen diluted 1:100 into fresh supplemented liquid medium and incubatedat 37° C., 250 rpm. For growth curve studies, 5 ml volumes wereperiodically removed from incubating cultures, from which bacteria from4 ml were pelleted, while the remaining 1 ml volume was used to measurethe optical density at 600 nm (OD₆₀₀). Pelleted bacteria wereresuspended in 1 ml of PBS, and cells then diluted 1:1,000 in PBS priorflow analysis. Quantitation of GFPuv fluorescence was analyzed using aMoFlo Legacy flow cytometer/cell sorter system (Beckman Coulter) withthe argon laser exciting bacteria at 488 nm and emissions detected at525 nm. Forward versus side light scatter, measured with logarithmicamplifiers, was used to gate on bacteria. A minimum of 50,000 eventswere acquired from each sample at a collection rate of approximately3,500 events per second. The mean fluorescence intensity was determinedusing Summit software (Beckman Coulter). Background autofluorescence wasdetermined using the negative control S. Typhi vaccine strain CVD 910.

Results

Construction of CVD 910.

The attenuated vaccine candidate CVD 908-htrA, derived from Ty2 andcarrying deletions in aroC, aroD, and htrA, was previously constructedand proved to be safe and highly immunogenic in Phase 2 clinicaltrials.[13] Here, a new vaccine strain, CVD 910, was constructed thatcarries deletions in guaBA and htrA. The ΔaroC ΔaroD was replaced withthe single deletion ΔguaBA for two important reasons: 1) previous workby the inventors showed that ΔguaBA alone sufficiently attenuates Ty2,resulting in a live vector strain capable of eliciting impressivehumoral immunity to a plasmid-encoded foreign antigen using the murineintranasal model of immunogenicity;[14] and 2) transcriptional controlof the guaBA locus is controlled by growth rate, independent ofguanine-mediated repression,[15] allowing expression of properlyintegrated antigen expression cassettes to be increased as the livevectors grow in the host. In order to reduce the risk of reversion tovirulence by the unlikely acquisition of wild type guaBA genes, asecondary deletion of htrA which encodes a heat shock-induced serineprotease was further engineered.

Deletion cassettes targeting guaBA and htrA were constructed for usewith the λ Red-mediated site-directed mutagenesis method,[16] and eachcassette was used to successfully delete either guaBA or htrA fromwildtype S. Typhi Ty2. Introduction of both deletion mutations into asingle strain resulted in the creation of CVD 910. A preliminaryassessment of attenuation of CVD 910 was carried out by comparing theminimum lethal dose causing death in 50% of a group of BALB/c mice(LD50) for CVD 910 versus CVD 908-htrA, using the hog gastric mucinintraperitoneal murine challenge model. For this model, the guidelinesrecommended in the Code of Federal Regulations for Food and Drugs, Title21, Part 620.13 (c-d), 1986 for intraperitoneal challenge of mice withS. Typhi were broadly followed. Using this method, the LD50 for both CVD910 and CVD 908-htrA was determined to be approximately 5×10⁵ CFU (datanot shown), versus an LD50 of ˜10 CFU for wild-type Ty2,[17]demonstrating construction of a novel live vaccine strain with a safetyprofile equivalent to that of CVD 908-htrA.

Chromosomal Integration of Gfpuv Cassettes into CVD 910.

GFPuv was expressed from independently controlled cassettes in CVD 910(containing the guaBA and htrA chromosomal gene deletions) in thefollowing manner. The osmotically regulated P_(ompC) promoter wasgenetically fused to a promoterless gfpuv gene. The resultingP_(ompC)-gfpuv cassette was integrated into either the guaBA or htrAloci such that only the open reading frame was replaced, but theoriginal promoters for both chromosomal loci were preserved, as depictedschematically in FIG. 1. For example, integration of P_(ompC)-gfpuv intothe guaBA locus to create CVD 910-GG resulted in transcription of gfpuvcontrolled both by osmolarity (via P_(ompC)) and growth rate (viaP_(guaBA)). Similarly, integration of the same cassette into htrA tocreate CVD 910-HG resulted in synthesis of GFPuv controlled both byosmolarity (P_(ompC)) and heat shock/environmental stress(P_(htrA)).[18] In addition, a third chromosomal integration wasprepared, CVD 910-CG, in which P_(ompC)-gfpuv replaced clyA, encoding acryptic hemolysin from Ty2 whose transcription is normally controlled bylow pH.[19] Interestingly, when the resulting strains were grownovernight at 37° C. in liquid cultures and analyzed for fluorescence byflow cytometry, observed fluorescence intensity was found to be stronglyinfluenced by the site of integration, regardless of osmotic inductionof P_(ompC). As shown in the fluorescence histograms of FIG. 2, underinducing conditions of 200 mM NaCl, strains with P_(ompC)-gfpuvintegrated into either guaBA or htrA displayed remarkably uniformbacterial populations with mean fluorescence intensities of 28.65 and21.59 respectively, while integration into clyA resulted in a very lowmean fluorescence intensity of 7.53, barely above the backgroundautofluorescence of 5.94 detected for CVD 910 alone. Having establishedsubstantial expression of GFPuv from two independent chromosomal loci,the hypothesis that integration of P_(ompC)-gfpuv into both guaBA andhtrA together would result in additive expression of fluorescence wasthen tested. Analysis of fluorescence from the resulting strain, CVD910-2G, revealed an uninduced (50 mM NaCl) mean fluorescence intensityof 36.01, which increased to 48.21 after induction with 200 mM NaCl. Inthis experiment, uninduced fluorescence intensities for CVD 910-GG andCVD 910-HG were 25.35 and 15.85 respectively, while induced fluorescencelevels were 32.46 and 24.03 respectively. It is immediately evident thatfor overnight liquid cultures, cumulative fluorescence observed with twocopies of gfpuv integrated into CVD 910-2G is approximately equivalentto the combined fluorescence levels for individual copies of integratedgfpuv observed in CVD 910-GG and CVD 910-HG, under both uninduced andinduced osmotic conditions.

Growth-Phase Regulated Expression of GFPuv in CVD 910-2G.

Regulated, but sustained, expression of foreign antigens delivered bylive vectors is expected to reduce any metabolic burden associated withantigen synthesis, thereby allowing live vectors to persist longer inimmunized hosts and prolong delivery of candidate vaccine antigens tothe immune system. [20] However, and despite recent improvements,tightly regulated and appropriately timed antigen expression usingplasmid-based expression technologies still remains elusive in manycases, with leaky expression potentially contributing toover-attenuation of live vector vaccine strains. Therefore, one of thegoals of the current work was to investigate the feasibility of linkingforeign antigen expression to the growth phase of the live vector, suchthat expression would be reduced when bacteria are adapting to asignificant change in environmental conditions (i.e. lag phase), butwould be strongly induced after bacteria have successfully adapted theirmetabolism to new energy sources and environmental conditions (i.e.exponential growth transitioning into stationary phase).

To meet this goal, chromosomally-encoded GFPuv expression in CVD 910-2Gwas first compared to a previously described live vector CVD908-htrAssb(pGEN206) [3], in which GFPuv was expressed independently ofgrowth phase from a low copy (˜5 copies per chromosomal equivalent)stabilized expression plasmid. Overnight starter cultures of CVD 910-2Gand CVD 908-htrAssb(pGEN206) were grown at 37° C. for approximately 16hrs and then diluted 1:100 into 100 ml of fresh medium in 250 ml baffleflasks. To reduce the influence of osmolarity on growth phase and moreclearly establish any link between observed fluorescence and inductionof P_(guaBA) and P_(htrA) during growth, all strains were grown undernon-inducing conditions of 50 mM NaCl. Fresh cultures were incubated at37° C./250 rpm, and 5 ml aliquots were removed every hour for 6 hours tomeasure both OD₆₀₀ and fluorescence intensities by flow cytometry. Asexpected, plasmid-based expression in CVD 908-htrAssb(pGEN206)significantly slowed the growth kinetics of the live vector whencompared to either CVD 910 or CVD 910-2G, even under non-inducingconditions of 50 mM NaCl (Table 2). Initial fluorescence intensities inlag phase started out quite high at 1262.66, dipped during exponentialphase to 686.27, and then rose again to 1131.59 in stationary phase. Insharp contrast, the kinetics of GFPuv expression in CVD 910-2G wasclosely linked to the growth phase of the culture, with a low meanfluorescence intensity of 81.19 measured in the lag phase, whichgradually increased with cell density to a maximum fluorescenceintensity of 200.06 as the culture reached stationary phase. Theobserved variation of fluorescence with growth phase, as quantitated byflow cytometry, is not an aggregate effect of increasing cell numbers,but instead reflects the level of GFPuv synthesis within individualbacteria in a growth-rate dependent manner. These data support thefeasibility of chromosomal expression of a foreign antigen from multipleintegration sites, and the possibility of antigen expressionsynchronized with growth-rate, a possibility not supported byplasmid-based expression in these experiments.

TABLE 2 Chromosomal versus plasmid-based expression of GFPuv inattenuated Salmonella Typhi live vectors. CVD 910-2G CVD 908htrAssb TimeCVD 910 (guaBA::gfpuv htrA::gfpuv) (pGEN206S2) (hr) OD₆₀₀ ^(a) MFI^(b)OD₆₀₀ MFI OD₆₀₀ MFI 0 0.04  ND^(c) 0.04 ND 0.04 ND 1 0.07 ND 0.08 81.190.06 1262.66 2 0.27 ND 0.3 96.77 0.14 1196.59 3 0.71 ND 0.71 105.59 0.38721.34 4 1.36 ND 1.36 182.77 0.72 686.27 5 1.88 ND 1.86 ND 1.25 ND 62.18 6.34 2.18 169.87 1.67 891.53 7 2.29 ND 2.29 200.06 1.95 1131.59^(a)Cultures grown under non-inducing conditions in 50 mM NaCl. ^(b)MeanFluorescence Intensity. ^(c)Not Determined.

This experiment was repeated to compare GFPuv expression from doubleintegrations in CVD 910-2G to single integration expression levels inCVD 910-GG and CVD 910-HG. As summarized in Table 3, growthphase-dependent expression of fluorescence intensity was again observed,increasing from an initial lag phase level of 32.90 to a high of 161.65in stationary phase. Interestingly, fluorescence levels during the 3 hrlag phase for the double integration did not reflect the sum offluorescence observed with single integrations during this period, butbecame additive as the cultures progressed into exponential andstationary phases. Fluorescence intensities from single integrations didnot seem to reflect the same dependence on growth phase as observed forthe double integration; intensities for the guaBA integration in CVD910-GG progressed from 74.94 to 96.31 during growth whilehtrA-controlled fluorescence in CVD 910-HG progressed from 32.90 to68.94. Despite this anomaly, the data reported here suggest thatintegration of antigen expression cassettes into multiple loci within alive vector chromosome can be accomplished without further attenuationof the vaccine strain, and that this multiple integration strategyresults in superior expression levels of foreign antigens versusconventional integration into a single locus.

TABLE 3 Growth-phase regulated chromosomal expression of GFPuv in CVD910 attenuated Salmonella Typhi live vectors. CVD 910-2G CVD 910-GG CVD910-HG (guaBA::gfpuv Time CVD 910 (guaBA::gfpuv) (htrA::gfpuv)htrA::gfpuv) (hr) OD₆₀₀ ^(a) MFI^(b) OD₆₀₀ MFI OD₆₀₀ MFI OD₆₀₀ MFI 00.04  ND^(c) 0.04 ND 0.03 ND 0.02 ND 1 0.09 ND 0.09 74.94 0.06 32.9 0.0638.31 2 0.33 ND 0.3 71.03 0.24 49.08 0.24 72.53 3 0.81 ND 0.72 70.580.68 56.12 0.6 95.41 4 1.45 ND 1.31 75.26 1.29 60 1.36 121.95 5 1.96 ND1.86 84.81 1.84 66.55 1.86 138.01 6 2.24 5.87 2.17 96.31 2.19 68.94 2.16161.65 ^(a)Cultures grown under non-inducing conditions in 50 mM NaCl.^(b)Mean Fluorescence Intensity. ^(c)Not Determined.

Construction and Testing of CVD 910-3A.

An additional strain of CVD 910 was prepared that expresses the cellbinding domains from toxin A (CBD/A) or from toxin B (CBD/B) of C.difficile. A synthetic codon-optimized gene cassette encoding the cellbinding domain from toxin A designated 14cbd/a was prepared where theosmotically regulated P_(ompC) promoter was genetically fused to apromoterless 14cbd/a gene. All P_(ompC)-controlled antigen cassettesencoding C. difficile antigens were constructed by inserting syntheticcodon-optimized genes (encoding the cell binding domains of either14CBD/A (SEQ ID NO:23) or CBD/B (SEQ ID NO:24)) as NheI-AvrII fragmentsinto pSEC10 digested either with SpeI-NheI to generate the unfusedP_(ompC)-14cbd/a encoding 14CBD/A, or pSEC10 cleaved only with NheI togenerate the fused P_(ompC)-clyA::cbd/b encoding ClyA-CBD/B. In the caseof P_(ompC)-14cbd/a, the resulting cassette was then excised from pSEC10as an EcoRI-AvrII fragment and inserted into chromosomal integrationcassettes in preparation for crossing into the chromosome usingpreviously published λ Red integration technologies (see FIG. 3) [3,16].All integration cassettes were integrated such that only the openreading frame of either guaBA or htrA was replaced, but the originalpromoters for both chromosomal loci were preserved, as depictedschematically in the chromosomal integration strategy of FIG. 3. Forexample, integration of P_(ompC)-14cbd/a into the guaBA locus resultedin transcription of 14CBD/A controlled both by osmolarity (via P_(ompC))and growth rate (via P_(guaBA)). Similarly, integration of the samecassette into htrA resulted in synthesis of 14CBD/A antigen controlledboth by osmolarity (P_(ompC)) and heat shock/environmental stress(P_(hfrA)).[18]

In addition, advantage was taken of the fact that all strains derivedfrom Ty2 are naturally inactivated at the rpoS locus [24] in order tointegrate a third copy of P_(ompC)-14cbd/a into the chromosome of CVD910 without further attenuation of the live vector vaccine. Integrationof P_(ompC)-14cbd/a into rpoS resulted in expression of 14CBD/A antigencontrolled by osmolarity (P_(ompC)) and entry of growing vaccineorganisms into stationary phase (P_(rpoS)) [25]. Additional primers usedto construct the rpoS-targeted integration cassettes are listed below inTable 4.

TABLE 4Primers used in the construction and testing of live vector strains expressingchromosomally encoded 14CBD/A from the rpoS locus. Primer (SEQ ID NO:)Sequence^(a) 5rpoS-for5′- AAGCTTGAATTCCGTATTCTGAGGGCTCAGGTGAACAAAGTGC -3′ SEQ ID NO: 255rpoS-rev 5′- CCTAGGCAATTGACCCGTGATCCCTTGACGGAACTAGCAAGTC -3′SEQ ID NO: 26 3rpoS-for 5′- GGATCCGGTTCGGTATCGCGCCAGGTATACAGACAATGC -3′SEQ ID NO: 27 3rpoS-rev 5′- CTCGAGCCGGAAGTGCAGGCGGTAAACGCTATGTACAC -3′SEQ ID NO: 28 rpoS PCR-for 5′- ATGCAGCACAGCAAGGAGTTGTGACCA -3′SEQ ID NO: 29 rpoS PCR-rev 5′- GGTGCGTATCGATAAGGTCTCTTACCACAGC -3′SEQ ID NO: 30 ^(a)Relevant restriction sites are underlined.

Successful integration of P_(ompC)-14cbd/a into guaBA, htrA, and rpoS,creating the live vector strain CVD 910-3A, was verified by directchromosomal sequencing and listed here as SEQ ID NO:31, SEQ ID NO:32,and SEQ ID NO:33; the protein amino acid sequence for 14CBD/A is listedas SEQ ID NO:34. In all chromosomal sequences presented, the location ofthe P_(ompC) promoter region, sequences encoding 14CBD/A, and residualFRT chromosomal scar sequences (left behind after removal of thekanamycin resistance marker) shown in Table 5. The location of keyrestriction sites (BamHI: GGATCC and XbaI: TCTAGA) are also shown in theTable as points of reference to be related back to the chromosomalintegration strategy shown in FIG. 3.

TABLE 5 P_(ompC) Antigen residual FRT BamHI: XbaI: NheI: promoter codingchromosomal GGATCC TCTAGA GCTAGC region region scar sequences site sitesite SEQ ID 10-984 991-996 13-18 NO: 23 14CBD/A SEQ ID 10-1617 1624-162913-18 NO: 24 CBD/B SEQ ID 876-1361 1388-2362 2437-2470  1362-1367;2152-2157 NO: 31 14CBD/A 2486-2491 SEQ ID 830-1315 1342-2316 2391-2424 1316-1321; 2406-2411 NO: 32 14CBD/A 2440-2445 SEQ ID 655-1140 1167-21412231-2264  1141-1146; 2246-2251 NO: 33 14CBD/A 2280-2285 SEQ ID 1-325NO: 34 14CBD/A SEQ ID 1431-3029 699-732 714-719 NO: 35 14CBD/A SEQ ID 1-489 1431-3029  516-1430  490-495; 1425-1430 NO: 36 14CBD/A 3084-3089SEQ ID 306-838  1-305 NO: 37 14CBD/A

Copy number-dependent osmotically controlled expression of 14CBD/A wasconfirmed by western immunoblot analysis. As shown in FIG. 4, six hourliquid broth cultures of CVD 910-2A (carrying P_(ompC)-14cbd/aintegrated into guaBA and htrA) were compared to cultures of CVD 910-3A(carrying P_(ompC)-14cbd/a integrated into guaBA, htrA, and rpoS). Allcultures were grown at 37° C. under either inducing (200 mM NaCl toactivate P_(ompC)) or non-inducing (15 mM NaCl) conditions. Induction of14CBD/A synthesis is clearly observed, with maximum expression confirmedfor CVD 910-3A induced with high osmolarity.

Construction of CVD 910-3Assb(pSEC10-CBD/B).

A chromosomal deletion of ssb was introduced into CVD 910-3A aspreviously described [3], accompanied by introduction of thenon-antibiotic SSB-stabilized expression plasmid pSEC10 into which asynthetic codon-optimized gene cassette encoding the cell binding domainof C. difficile toxin B was inserted. The resulting live vaccine strain,designated CVD 910-3Assb(pSEC10-CBD/B) is depicted schematically in FIG.5. Confirmation of the chromosomal deletion of ssb as intended wasconfirmed by direct chromosomal sequencing as listed in SEQ ID NO:35;the integrity of the plasmid-based P_(ompC)-clyA-cbd/b cassette was alsoconfirmed by direct sequencing as listed in SEQ ID NO:36, with thepredicted amino acid sequence of ClyA-CBD/B listed in SEQ ID NO:37. Hereagain, for SEQ ID NO:35, the location of the residual FRT chromosomalscar sequences (replacing the deleted ssb gene) is shown in Table 5along with the location of the internal XbaI site (TCTAGA). For the SEQID NO:36 sequence encoding ClyA-CBD/B, the location of the P_(ompC)promoter region is also shown in Table 5 along with the locations of thesequence encoding CBD/B and the key restriction sites (BamHI: GGATCC,NheI: GCTAGC, and AvrII: CCTAGG).

Proof-of-Principle Immunogenicity and Challenge Experiment Using a CVD910 Bivalent Plague Vaccine.

The strategy for development of CVD 910-3Assb(pSEC10-CBD/B) was informedby a critical proof-of-principle experiment in which a bivalent livevector vaccine against pulmonary plague caused by Yersinia pestis wasconstructed and tested. Using the identical genetic engineering strategyused to create CVD 910-3Assb(pSEC10-CBD/B), a bivalent CVD 910-basedplague vaccine was constructed that expressed the full-length LcrVantigen (required for secretion of virulence effectors proteins and avirulence factor by itself) from the three independent guaBA, htrA, andrpoS chromosomal sites, each containing an osmotically-regulatedP_(ompC)-lcrV cassette. The protective anti-phagocytic capsular F1antigen was expressed from the SSB-stabilized non-antibiotic low copyexpression plasmid pSEC10, creating the plasmid pSL445. The F1 antigenof pSL445 was encoded by the natural Y. pestis caf1 operon butengineered to be transcriptionally controlled by the in vivo-induciblesifA promoter (an S. Typhi promoter controlled by the SalmonellaPathogenicity Island 2 (SPI 2) regulon), after having determined thatexpression of caf1 using the P_(ompC) promoter was toxic to CVD 910. Forcomparison, the bivalent plasmid pSL483 (again derived from pSEC10) wasalso constructed in which the expression of both the caf1 operon andlcrV were divergently transcribed from the P_(sifA) and P_(ompC)promoters respectively. SL483 was then introduced into CVD 908-htrAssbcreating a bivalent plague candidate vaccine CVD 908-htrAssb(pSL483) inwhich foreign antigen expression was completely plasmid encoded, to becompared to CVD 910-3Lssb(pSL445) in which foreign antigen expressionwas balanced between the chromosome and a plasmid.

The immunogenicity of these live vector vaccine strains was evaluatedusing a heterologous prime-boost immunization strategy in which BALB/cmice were primed intranasally with 1×10⁹ CFU of live vaccine on days 0and 28, followed by a boost with a small amount (500 nanograms) ofpurified lcrV adsorbed to alum on day 56. All mice were challenged onday 84 (i.e., 28 days after the last immunization) with 177 LD50s ofvirulent Y. pestis strain CO92. Results are presented in Table 6.

TABLE 6 Immunogenicity of S. Typhi live vector candidate plague vaccinesexpressing LcrV and F1 and further tested for efficacy in a lethalpulmonary challenge model. Day 28 Day 56 Day 84 Percent survival (before(before (4 weeks (14 days Vaccine boost 1) Day 42 boost 2) post boost 2)post challenge) F1-specific serum IgG CVD 910 12.5 12.5 12.5 12.5 40%CVD 910-3L 212.5 12.5 12.5 12.5 70% CVD 910-3Lssb(pSL445) 2,268.333,810.9 16,613.2 21,778.3 100%  CVD 908-htrAssb(pSL483) 445.2 11,056.11,706.5 3,684.7 100%  PBS prime-LcrV boost 12.5 12.5 12.5 12.5 20% PBS12.5 12.5 12.5 12.5  0% LcrV-specific serum IgG CVD 910 49.4 125.0 12.578,994.1 CVD 910FL 93.9 224.6 12.5 86,968.7 CVD 910-3Lssb(pSL445) 25.075.4 12.5 228,230.1 CVD 908-htrAssb(pSL483) 25.0 20,008.9 21,267.3407,085.8 PBS prime-LcrV boost 25.0 51.3 12.5 28,855.7 PBS 25.0 25.012.5 12.5 Typhi LPS-specific serum IgG CVD 910 168.5 3,570.8 Pending30,192.9 CVD 910-3L 311.8 7,088.0 18,754.0 52,266.0 CVD910-3Lssb(pSL445) 171.5 1,366.9 Pending 18,917.4 CVD 908-htrAssb(pSL483)135.3 1,248.5 Pending 1,968.6 PBS prime-LcrV boost 157.8 121.3 307.0244.2 PBS 150.9 116.5 297.6 188.7

These results clearly show that when expression of foreign antigens isbalanced between inducible multilocus chromosomal expression andinducible plasmid-based expression, serum antibody responses againstboth foreign antigens LcrV and F1 were equivalent to that observed whenboth antigens were expressed from a single stabilized expressionplasmid. Perhaps more importantly, when examining live vector-specificLPS responses, serum IgG responses 10 fold higher were observed in miceimmunized with CVD 910-3Lssb(pSL445) versus responses in mice immunizedwith CVD 908-htrAssb(pSL483) (day 84 GMT=18,917.4 versus 1,968.6respectively). These results strongly support the hypothesis that themetabolic burden associated with expression of multiple foreign antigensin attenuated multivalent live vector vaccines can be reduced or eveneliminated by engineering appropriately balanced levels of antigenexpression, accomplished by strategic distribution of foreign genesbetween multiple chromosomal loci and genetically stabilized low copyplasmids.

Construction of a Multivalent CVD 910 Live Vector Vaccine TargetingEnterotoxins and a Putative Colonization Factor of C. difficile.

Enterotoxins A (TcdA) and B (TcdB) are the primary virulence factors ofC. difficile. However, epidemic strains of C. difficile invariably carryan additional toxin affecting the actin cytoskeleton of intestinal cellscalled C. difficile transferase (Cdt); this toxin has also been calledbinary toxin (BT) because it is composed of a catalytic A subunit and acell-binding B subunit (30, 31). The activity of BT causes rearrangementof the actin cytoskeleton of intestinal epithelial cells, disruptingtight junctions and potentially allowing better penetration ofenterotoxins into gastrointestinal tissue (32), thereby enhancing thevirulence of epidemic strains. It was recently discovered that BT alsoappears to enhance colonization of the intestinal tract by inducingmicrotubule-based protrusions which enhance the adherence of C.difficile (33). Based on available data, it was hypothesized that binarytoxin acts to enhance the virulence of epidemic strains carrying allthree toxins by promoting better colonization of C. difficile andpossibly improving the penetration and binding of enterotoxins A and B.Recent animal studies suggest that immunization against enterotoxinsalone does not prevent colonization of the gastrointestinal tract by C.difficile (34), and that strains producing only binary toxin are able tocolonize susceptible animals (35). Therefore, live vector-mediatedmucosal immunity against C. difficile disease will be targeted at threelevels: 1] by blocking the binding of both enterotoxins throughtargeting of serum immunity to their cell-binding domains, 2] byinducing mucosal immunity to the cell binding domain of BT (designatedhere as CBD-BT) to reduce penetration of toxins A and B by maintainingthe integrity of intestinal epithelial tissue, and 3] by targetingmucosal immunity against CBD-BT to reduce intestinal colonization,recurrent infection, and environmental transmission in a clinicalsetting.

Towards this goal of constructing a trivalent live vaccine against C.difficile infections in which mucosal immunity is targeted againstenterotoxins A, B, and binary toxin, CVD 910-3A was first modified toexpress CBD/A from one further chromosomal locus, namely the clyA locus.This modification serves to avoid loss of expression of chromosomallyencoded 14CBD/A from the three chromosomal loci of CVD 910-3A.Integration of P_(ompC)-14cbd/a into the clyA locus was completed usingthe method described above in the paragraph entitled “Construction andtesting of CVD 910-3A”; this integration cassette was integrated suchthat only the open reading frame encoded by clyA was replaced, whilepreserving the original clyA promoter, as depicted schematically in thechromosomal integration strategy of FIG. 3.

The resulting monovalent vaccine strain, CVD 910-4A, was then furthermodified to contain and express CBD-BT fused to the carboxyl terminus ofClyA in the manner routinely used in low copy number SSB-stabilizedexpression plasmids; as with other expression cassettes constructed inthese plasmids and later moved into chromosomal integration modules,this clyA::cbd-bt gene fusion was again transcriptionally controlled bythe osmotically regulated promoter P_(ompC). In order to take advantageof the previously constructed integration modules in which incomingexpression cassettes encoding foreign antigens were inserted asEcoRI-AvrII fragments, it was necessary to create a new synthetic geneencoding ClyA in which the internal naturally occurring EcoRI site wasremoved; the resulting synthetic gene (designated clyA*) was thenre-inserted into pSEC10 to create pSEC10S2 (SEQ ID NO:45). In additionto the EcoRI site (base 1034, T to C), other commonly used restrictionsites in pSEC10S2 (SEQ ID NO:45) were removed, including BglII (base590, T to C), two HindIII sites (base 686, A to G; base 1043, A to G),HpaI (base 978, T to C; base 980, A to G) and MfeI (base 1262, A to G).

A synthetic 2655 bp codon-optimized gene encoding the 878 residues ofCBD-BT (SEQ ID NO:46) was then synthesized as a NheI-AwII fragment (SEQID NO:47). However, insertion of this cassette into pSEC10S2 cleavedwith NheI was unsuccessful. A smaller gene cassette encoding residues212-878 of CBD-BT (designated B2; SEQ ID NO:46) was amplified using theforward primer5′-AGATCTaaaataaggaggaaaaaaaaATGGCTAGCCTGATGTCTGATTGGGAAGATGAAG-3′ (NheIsite underlined; SEQ ID NO:51) and the reverse primer5-AAGCTTCCTAGGTTATTAATCCACACTCAGAACCAGCAGTTCG-3′ (AvrII site underlined;SEQ ID NO:52); this decision was based on a report by Sundriyal et al.(36) demonstrating the biological activity of this truncated portion ofCBD-BT which results naturally from proteolytic cleavage of thefull-length 98.8 kDa CBD-BT, removing a 20 kDa N-terminal fragment, andresulting in a soluble 74.9 kDa protein. The resulting 2054 bp syntheticgene (SEQ ID NO:48) was then successfully inserted as a NheI-AvrIIfragment into pSEC10S2 cleaved with NheI to create pSEC10S2-B2 encodinga 972 residue 108.6 kDa ClyA-CBD-BT fusion protein (SEQ ID NO:49). Thisplasmid was then introduced into CVD 910-4A to create the bivalentvaccine CVD 910-4Assb(pSEC10S2-B2).

The P_(ompC)-clyA*-b2 expression cassette was then excised frompSEC10S2-B2 as a 3491 bp EcoRI-AvrII fragment (SEQ ID NO:50), insertedinto the guaBA integration module cleaved with MfeI-NheI, and thenintegrated into the guaBA locus of CVD 910-4A, replacing the previouslyintegrated P_(ompC)-14cbd/a cassette to create the bivalent live vaccineCVD 910-3A-GB2 in which 14CBD/A is chromosomally expressed from thehtrA, rpoS, and clyA loci, and CBD-BT is expressed from the guaBA locus.When export of the protein fusion was compared for hemolytic activitywith plasmid-encoded fusion protein expressed in CVD910-4Assb(pSEC10S2-B2), proper export of the fusion protein was observedin both strains, although plasmid-encoded export was much higher due tocopy number (FIG. 6).

The SSB-stabilized plasmid encoding the cell binding domain ofenterotoxin B, pSEC10-CBD/B, is introduced into CVD 910-3A-GB2 to createthe trivalent live vaccine CVD 910-3A-GB2ssb(pSEC10-CBD/B) shown in FIG.7 in which 14CBD/A is chromosomally expressed from the htrA, rpoS, andclyA loci, CBD-B2 is expressed from the guaBA locus, and CBD/B isexpressed from pSEC10-CBD/B.

Alternatively, the SSB-stabilized plasmid encoding the cell bindingdomain of enterotoxin B, pSEC10-CBD/B, is introduced into CVD910-2A-GRB2 to create the trivalent live vaccine CVD910-2A-GRB2ssb(pSEC10-CBD/B) in which 14CBD/A is chromosomally expressedfrom the htrA and clyA loci, CBD-B2 is expressed from the guaBA locusand rpoS locus, and CBD/B is expressed from pSEC10-CBD/B.

To improve binary toxin-specific toxin neutralizing activity, a secondcopy of P_(ompC)-clyA*-b2 can be inserted into the rpoS locus of CVD910-3A-GB2ssb(pSEC10-CBD/B).

While the invention has been described with reference to certainparticular embodiments thereof, those skilled in the art will appreciatethat various modifications may be made without departing from the spiritand scope of the invention. The scope of the appended claims is not tobe limited to the specific embodiments described.

REFERENCES

All patents and publications mentioned in this specification areindicative of the level of skill of those skilled in the art to whichthe invention pertains. Each cited patent and publication isincorporated herein by reference in its entirety. All of the followingreferences have been cited in this application:

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What is claimed is:
 1. An attenuated strain of Salmonella entericaserovar Typhi (“S. Typhi”) having disruptions in four or morechromosomal locations, said chromosomal locations comprising the guaBAlocus, the htrA locus, the rpoS locus, and the ssb locus.
 2. Theattenuated strain of claim 1, wherein the attenuated strain furthercomprises a chromosomal-based expression system integrated into at leastone location of chromosomal disruption, wherein each chromosomal-basedexpression system comprises an expression cassette encoding an antigen.3. The attenuated strain of claim 2, wherein the attenuated straincomprises at least two chromosomal-based expression systems.
 4. Theattenuated strain of claim 3, wherein the attenuated strain comprisestwo different chromosomal-based expression systems.
 5. The attenuatedstrain of claim 2, wherein the attenuated strain comprises at leastthree chromosomal-based expression systems.
 6. The attenuated strain ofclaim 5, wherein the attenuated strain comprises three differentchromosomal-based expression systems.
 7. The attenuated strain of claim2, wherein the attenuated strain comprises at least fourchromosomal-based expression systems.
 8. The attenuated strain of claim7, wherein the attenuated strain comprises four differentchromosomal-based expression systems.
 9. The attenuated strain of claim2, wherein the attenuated strain further comprises one or moreplasmid-based expression systems, and wherein each plasmid-basedexpression system comprises an expression cassette encoding an antigen.10. The attenuated strain of claim 9, wherein the plasmid-basedexpression system is an SSB-stabilized plasmid-based expression system.11. The attenuated strain of claim 2 formulated as a pharmaceuticalcomposition comprising a pharmaceutically-acceptable carrier or diluent.12. A method of inducing an immune response to an antigen in a subject,comprising administering to the subject a pharmaceutical compositionaccording to claim
 11. 13. The attenuated strain of claim 9 formulatedas a pharmaceutical composition comprising a pharmaceutically-acceptablecarrier or diluent.
 14. A method of inducing an immune response to anantigen in a subject, comprising administering to the subject apharmaceutical composition according to claim 13.